PROJECT SUMMARY CD4+ regulatory T cells (Tregs) are essential for normal immune surveillance, and their dysfunction can lead the development of autoimmune diseases, such as rheumatoid arthritis. Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source of healthy Tregs to treat autoimmune arthritis as they have the ability to produce almost all cell types in the body, including Tregs. However, the right conditions for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) have not been fully defined, especially the signaling mechanisms that direct differentiation of such Tregs. Ag-specific PSC-Tregs can be tissue-associated and infiltrate to local inflamed tissue (e.g., synovium) to suppress autoimmune responses after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. The long-term goal of our research program is to develop and optimize strategies for utilizing PSCs as a source of highly reactive Tregs for cell-based therapies. The objective of this application is to determine the mechanisms underlying the Ag- specific PSC-Treg treatments that aim to modulate tolerance in autoimmune arthritis. The central hypothesis is that auto Ag-specific PSC-Tregs are tissue-associated Tregs, which accumulate in inflamed synovium and suppress the autoimmunity after adoptive transfer and act as excellent candidates for cell-based therapies. Guided by published results and preliminary data from the applicant's laboratory, this hypothesis will be tested by pursuing three specific aims: 1) to define the fundamental properties of auto Ag-specific PSC-Tregs that relate to suppressive activity; 2) to determine the signaling mechanisms that direct the differentiation of auto Ag-specific PSC-Tregs; and 3) to study cell-based therapies of autoimmune arthritis using auto Ag-specific PSC- Tregs. Under the first aim, in vitro and in vivo approaches, which have been established as feasible in the applicant's laboratory, will be used. The fundamental properties of auto Ag-specific PSC-Tregs with regard to phenotype, specificity and function will be defined. Under the second aim, gene expression profile and intracellular signaling pathways will be studied to determine the critical roles of Notch-Hes1, Notch-Runx1 and Notch-survivin signaling during the development of auto Ag-specific PSC-Tregs. Under the third aim, well- characterized animal models will be used to optimize cell-based therapies of autoimmune arthritis using auto Ag-specific mouse or human PSC-Tregs. The approach is innovative, because the concept of auto Ag-specific PSC-Tregs for cell-based therapies in autoimmune arthritis has not been previously explored. The proposed research is significant, because it will provide the foundation for developing tissue-associated PSC-Tregs that will reduce overall immunosuppression after adoptive transfer by accumulating inflamed synovium and drive forward the use of therapeutic PSC-Tregs for cell-based therapies in autoimmune arthritis.